The effect of KIR-blocking for HLA-C in SarsCov2 immune responses

Introduction

EMILIE:

The major histocompatibility complex (MHC), or HLA in humans, are known well-known for their crucial role in inducing effective immune responses against antigens. While both HLA-A and -B have been widely studied for their interaction with T-cells and T-cell receptors (TCRs), much less is known about HLA-C - possibly due to the overall lower expression of HLA-C molecules.

However, newly published research has shown that HLA-C may pose a larger role in the immune response than first expected; an unusually high number of HLA-C recognition in CD8+ T cells are now challenging the prior assumptions of TCR-dependent responses. The motivation for this study was to be able to distinguish between TCR-dependent and independent responses and hereby characterize TCR-specific and unspecific bindings of HLA-C molecules.

Overall the study found that another receptor, the killer immunoglobin receptors (KIR), seemed to block the binding of TCR to pHLA-C. Unlike the HLA-A and -B, the HLA-C alleles express proteins that are recognised by KIRs. These receptors are present (together with the TCR) on NK-cells and can be either inhibiting or activating.

Materials and Methods - Project Flow Chart

Materials and Methods

Data:

High-throughput experiment
Thousands of SarsCov2 peptides binding to T-cells
TCR-dependent or TCR-independent (i.e.: KIR-mediated)?

3 cohorts:

AP: Leukemia patients who have had COVID-19 (n = 12)
BS: Healthy nurses who have had COVID-19 (n = 17)
BC: Healthy donors (n = 15)

Methods:

DNA-barcoding: Tagging each peptide-HLA pair uniquely
Flow cytometry: Sorting T-cells based on TCR vs KIR binding

Materials and Methods - Barracoda data

	AP (N=13465)	BS (N=7840)	BC (N=10491)	Overall (N=31796)
KIR_blocking
1	7541 (56.0%)	4019 (51.3%)	7877 (75.1%)	19437 (61.1%)
0	5924 (44.0%)	3821 (48.7%)	2614 (24.9%)	12359 (38.9%)
log_fold_change
Mean (SD)	-1.15 (3.29)	-2.25 (3.95)	-1.49 (3.69)	-1.53 (3.62)
Median [Min, Max]	-0.182 [-13.4, 9.71]	-0.508 [-12.0, 8.64]	-0.313 [-12.4, 11.2]	-0.290 [-13.4, 11.2]
protein
ORF1	7239 (53.8%)	4447 (56.7%)	6103 (58.2%)	17789 (55.9%)
Nucleocapsid	707 (5.3%)	434 (5.5%)	548 (5.2%)	1689 (5.3%)
Spike	2619 (19.5%)	1581 (20.2%)	2062 (19.7%)	6262 (19.7%)
ORF6	68 (0.5%)	42 (0.5%)	65 (0.6%)	175 (0.6%)
Membrane	785 (5.8%)	465 (5.9%)	545 (5.2%)	1795 (5.6%)
ORF3	540 (4.0%)	345 (4.4%)	467 (4.5%)	1352 (4.3%)
ORF7	288 (2.1%)	175 (2.2%)	227 (2.2%)	690 (2.2%)
Envelope	119 (0.9%)	73 (0.9%)	91 (0.9%)	283 (0.9%)
ORF10	119 (0.9%)	73 (0.9%)	91 (0.9%)	283 (0.9%)
ORF8	322 (2.4%)	205 (2.6%)	292 (2.8%)	819 (2.6%)
Unknown	659 (4.9%)	0 (0%)	0 (0%)	659 (2.1%)
HLA
HLA-C04:01	300 (2.2%)	600 (7.7%)	2400 (22.9%)	3300 (10.4%)
HLA-C06:02	2695 (20.0%)	1086 (13.9%)	1164 (11.1%)	4945 (15.6%)
HLA-C07:01	4014 (29.8%)	2950 (37.6%)	2655 (25.3%)	9619 (30.3%)
HLA-C07:02	6456 (47.9%)	3204 (40.9%)	4272 (40.7%)	13932 (43.8%)

~ 32 000 bar-codes (patient-HLA-peptide combinations)

44 patients
4 HLA types
11 protein types

Materials and Methods - Flowjo data

Flow cytometry data

56 patients - not total overlap with Barracoda
Not all patients have been tested with and without KIR-blocking

	KIR-blocked (N=30)	Not KIR-blocked (N=26)	Overall (N=56)
APC_positive
Mean (SD)	0.682 (0.626)	1.43 (2.58)	1.03 (1.84)
Median [Min, Max]	0.450 [0, 2.20]	0.305 [0, 9.19]	0.360 [0, 9.19]
DP_positive
Mean (SD)	0.210 (0.376)	0.0319 (0.0905)	0.128 (0.294)
Median [Min, Max]	0.0510 [0, 1.61]	0 [0, 0.350]	0.0105 [0, 1.61]
PE_positive
Mean (SD)	2.86 (3.18)	6.32 (9.09)	4.46 (6.78)
Median [Min, Max]	2.18 [0.100, 17.2]	3.24 [0.160, 46.1]	2.44 [0.100, 46.1]

2 Fluorochromes:

PE: binding to SarsCov2
APC: binding to control peptides
DP = Double positive for APC and PE

Results - Filtering only significant responses

The number of observations reduced drastically during wrangling:

Original: 31796
w. Flowjo: 22819
Significant: 542

Results - Paired boxblot

Paired data to compare TCR-dependent and TCR-independent responses:

This further reduced the dataset size:

Original: 31796
Significant: 542
Paired: 22
- 11 pairs
  - 1 normal
  - 1 KIR-blocked

Results - Bubble plot - significant

\[ Est. Freq = \left( \frac{count_{barcode}}{count_{total}} \cdot 100 \% \right) \cdot PE \]

Results - Bubble plot - insignificant

\[ Est. Freq = \left( \frac{count_{barcode}}{count_{total}} \cdot 100 \% \right) \cdot PE \]

EMILIE:

The estimated frequency indicates the fraction of T cells that only recognises that specific peptide and was calculated earlier as: For C0401, only the BC cohort shows significant responses in both KIR blocked and non blocked samples. C0602, the BS cohort shows a significant decrease in the amount of significant responses after KIR blocking (lots of red dots, very few blue (obs maybe I changed the colors??)). This could also be caused by the fact that these samples are non paired, potentially making the donor variation affect the repertoire of significant responses. For C0702 and C0701 we see peptide specific responses that disappears after KIR blocking.

In general there is a tendency that KIR blocking is seemingly less effective than expected, although we do see a fall in responses for specific HLA C allotypes across the cohorts.

Discussion

KIR- blocking was showed to reduce the binding of pHMC
- suggesting TRC-independent binding
KIR-blocking was less efficient in reducing the binding of pHMC in the AP cohort
- hematological patients may have a lower KIR expression
Some results suggest that KIR-blocking is not as robust/reliable as expected

Limitations

The paired dataset was really small, hard to draw reliable conclusions
New info from this research group indicates that the KIR-antibody used in this experiment only blocked KIR-C1 but not -C2
- C0401 and C0601 are C2 ligands and were therefore not blocked
- This would explain the unreliable results, and variation between cohorts

MANON:

The registration of false positive responses were shown on a small subset of paired samples - it would require additional analysis in the future to confirm TCR specific response without the presence of non-blocked KIRs.

We could have made some changes to the threshold values to include more samples. We might lose some of the KIR blocked responses that falls beneath the threshold.

We have two sub types of KIR receptors, namely C1 and C2. However, just last week the phDs who made the dataset found that only C1 is blocked with the blocking antibody that we have. This means, that not all of them are blocked and some are still able to bind to pMHC. C0401 and C0601 are C2 ligands This makes us wonder whether all KIR receptors are truly blocked by the anti-KIR antibody used in this experiment